The venous system of E14.5 mouse embryos—reference data and examples for diagnosing malformations in embryos with gene deletions

Abstract Approximately one‐third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high‐quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high‐resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage‐specific referenced data.


| INTRODUC TI ON
About 2% of human newborns worldwide are diagnosed as being affected by a hereditary disease (Dolk et al., 2010;EUROCAT, 2018;Morris et al., 2018). In many cases, this has negative effects on health and life quality and often results in a dramatically reduced life span. The suffering of the affected individuals, together with the highly negative socioeconomic impact, emphasises the urgency of developing novel diagnostic and therapeutic strategies. The basis for such a development is profound knowledge of the effects genetic, biomechanical and environmental factors have on normal embryoand foetogenesis and peri-and postnatal development.
Luckily, gene function and basic developmental mechanisms are evolutionary conserved and a number of species, including drosophila, zebrafish, xenopus and the chick can be employed as models for investigating the triggers and driving factors of basal events in embryogenesis, tissue formation and remodelling. The most important model however is the mouse (Brown et al., 2018;Rosenthal & Brown, 2007). It is a species that is relatively closely related to humans, having a largely similar body plan. Furthermore, with a short reproduction time, a variety of tools for manipulating the mouse genome have been established. By analysing the phenotype of altered individuals, the effect of genetic, epigenetic or biomechanical factors on the formation and remodelling of tissue and organs can be explored. To facilitate the identification of phenotypic abnormalities, detailed descriptions of the normal anatomy and metric data of essential organ systems at important stages of intra-and extrauterine development have been made available (Captur et al., 2016;Desgrange et al., 2019;Geyer et al., 2017c;Weninger et al., 2009;Wong et al., 2014). Using them as a reference, they permit the diagnosis of phenotype abnormalities in genetically engineered or compromised individuals.
Systematic knockout efforts, initiated by the international mouse phenotyping consortium (IMPC, www.mouse pheno type.org), demonstrated that a third of the mouse genes are essential for embryo development and growth, since full deletion resulted in pre-or perinatal death of homozygous individuals (Ayadi et al., 2012;Dickinson et al., 2016). In these lines, careful phenotyping of embryos is the only possibility to diagnose the full spectrum of organ and tissue abnormalities linked to the function of the deleted gene. As identified in the program 'Deciphering the Mechanisms of Developmental Disorders' (DMDD), embryonic day (E)14.5 is the most important time point for such phenotype analyses (Mohun et al., 2013;Weninger et al., 2014, Wilson et al., 2016b. This is for several reasons: First, organogenesis is largely finished at E14.5, thereby permitting the detection of effects gene deletions have on the formation of all major organ systems relevant for embryo survival, growth and development; second, roughly half of the embryos of peri-or prenatally lethal mutant mouse lines survive until E14.5; third, a large proportion of embryos compromised by severe cardiovascular or endocrine malfunctions is still alive or at least not fully resorbed after intrauterine death and therefore still available for phenotype analysis (Geyer et al., 2017b;Mohun et al., 2013).
Another lesson learned from DMDD is that gene deletions in E14.5 embryos quite often result in subtle structural organ or tissue defects. The identification of such defects is challenging and requires the employment of cutting-edge three-dimensional (3D) imaging techniques. Since the high-resolution episcopic imaging (HREM) technique (Mohun & Weninger, 2011;Weninger et al., 2006) routinely provides digital volume data with voxel dimensions down to 3 × 3 × 3 µm 3 from whole E14.5 mouse embryos, DMDD and an increasing number of stand-alone projects employ this imaging method for phenotyping (Geyer & Weninger, 2019;Weninger et al., 2018). Its basis are series of two-dimensional (2D) digital images which nearly match the quality of images derived from glass slide mounted histological sections. However, HREM images are captured from subsequently exposed surfaces of a resin block during its sectioning on a microtome. Therefore, the single images do not show artefacts introduced by section processing and the full series of such images are precisely aligned, facilitating their simple conversion to a high-quality volume data.
Traditionally, phenotyping relies on direct comparisons of mutants with genetically normal littermates, harvested at the same time point and, ideally, stemming from the same dam. Recently, it was demonstrated that this approach is highly error prone, because developmental progress, size, morphology and topology of mouse embryos and their organs and tissues harvested at E14.5 can vary dramatically.
Comparing littermates without acknowledging stage differences and at the same time neglecting the spectrum of natural variation in the normal population is prone to cause false diagnosis of malformations and abnormalities. To overcome this problem, a staging system specifically for embryos harvested at E14.5 has been developed. It distinguishes 6 stages (S21, S22 − , S22, S22 + , S23 − and S23) of developmental progress, each showing several unique features, which might be considered as abnormal at other stages (Geyer et al., 2017b). Using this staging system, careful descriptions of the morphology and topology of the arterial system of E14.5 mouse embryos have recently been generated for each of the six identified stages.
These data have been successfully used as reference for diagnosing cardiovascular malformations in experimentally challenged and genetically engineered embryos (Geyer et al., 2017c). Although of similar importance for a comprehensive analysis of the cardiovascular system, comparable reference data defining the normal morphology of the venous system of E14.5 do not currently exist. We therefore decided to use careful 3D descriptions, analyses and visualisations of the venous anatomy of genetically normal E14.5 mouse embryos to provide such reference data, identifying the normal anatomical features of the venous system and exploring normal variations across the six successive stages of E14.5 development.

| MATERIAL S AND ME THODS
The phenotypes of 206 wild-type mouse embryos of the C57BL/6N strain and 56 mutants of 7 different knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c) were comprehensively analysed ( Table 1). All embryos were generated at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk) as part of the DMDD project (https://dmdd.org.uk) and its pilot, with embryos harvested at embryonic day (E) 14.5 and 13.5, respectively. The precise developmental stage of each embryo was subsequently determined using criteria based upon forelimb maturation (Geyer et al., 2017b).
Of the wild-type mouse embryos, 175 were harvested at E14.5 and 31 were harvested at E13.5. The latter were selected embryos of late E13.5 stages, which overlap in appearance with early E14.5 substages.
This increased the number of S21 embryos-a stage, which holds late E13.5, in addition to a small proportion of early E14.5 embryos.
Each series comprised a whole embryo. Since section thickness, and thus the distance between subsequent block face images ranged between 2.5 and 3.5 µm, the two-dimensional images were downscaled to gain isotropic voxel dimensions, before being virtually stacked. The virtual image stacks were converted to volume datasets and visualised employing Osirix Software (OsiriX v5.6, 64bit, Pixmeo Sarl), which ran on a MacPro Computer (3 GHz-8-Core Intel Xeon E5, 64 GB RAM, AMD FirePro D700). Using the straight and oblique section and 3D volume rendering tools, the data were carefully analysed following a detailed protocol for screening morphologic phenotypes of E14.5 embryos (Weninger et al., 2014). In 12 wild-type mouse embryos (Stages (S)21, 22−, 22, 22+, 23− and 23),

| RE SULTS
In general, veins do appear in HREM data as irregularly shaped or thin, slit-like structures. This is especially true for the umbilical and internal jugular vein and the inferior vena cava. In addition, the dimensions of corresponding veins differ significantly between individuals, even between individuals of identical E14.5 stages (S; Figure 2).

| Pulmonary veins
In all embryos, a single lung vein enters the left atrium from the dorsal side. It crosses over the left superior vena cava and opens into the left atrium immediately left to the septum primum. The single vein receives one vessel from the left and two from the right lung. The latter drain the right cranial, middle, caudal and accessory lobes ( Figure 2).

| Systemic veins
Three venae cavae enter the right atrium. One inferior vena cava enters from the caudal side. It starts with the connection of the two common iliac veins, ascends in the retroperitoneum, penetrates the diaphragm and runs for a relatively long distance inside the thorax, before it enters the heart.

| Intracranial venous system
All embryos show a forming superior sagittal and transverse sinus. The primitive superior sagittal sinus extends in the midline from anterior to the transition of fore-and midbrain. It receives TA B L E 1 Numbers of wild-type embryos analysed in this study

| Internal and external jugular and subclavian veins
The internal jugular vein starts at the jugular foramen, which is a large gap between cochlea and occipital bone in early embryos and an almost osseous canal in later embryos ( Figure 3).

| Umbilical vein and ductus venosus
In E14.5 embryos, large segments of the intestine are placed outside the embryo body and inside the physiological umbilical hernia.
Several venous channels enter the body through the umbilicus.
The umbilical vein is the dominant vessel. It runs between the layers of the wall of the umbilical hernia and enters the embryo body to the left of the colon. In a single embryo (0.5%), it entered the body to the right of the colon. Inside the body, it heads for the liver and enters the liver tissue at the porta hepatis, between the left and right lateral liver lobes. In 6% of embryos staged as S22+ and older, its diameter narrows at the level of the gall bladder.
Inside the liver, the diameter of the vessel enlarges significantly and it forms a voluminous, elongated and anteriorposteriorly orientated cavity. From this cavity branches emerge, which drain into all parts of the left medial liver lobe and the anteromedial part of the right medial liver lobe (Figure 4). At the posterior aspect of the cavernous part, the left branch of the portal vein joins and a very short ductus venosus starts, which connects to the inferior vena cava. In two-dimensional axial sections of 98% of the specimens, the connection appears as if secured by a slender valve. In 2%, this structure appears thickened and plump (Figure 4).

F I G U R E 4
Liver veins, portal venous system and umbilical vein. (a-h) Liver. Axial HREM-section (a), surface models of veins in context with axially sectioned semi-transparent volume model from cranial (b, c), coronal HREM re-section (e), and surface models in context with coronally sectioned semi-transparent volume models of the liver (f-h). Vena cava inferior (blue), portal venous system (light blue), vitelline vein (light blue), superior mesenteric vein (light blue), umbilical vein and branches (magenta), branches of left liver vein (yellow), middle liver vein (turquoise), right liver vein (green), caudal liver vein (red). Note that the branches of the umbilical vein feed mainly the left medial and parts of the right medial liver lobe. (i, j) Ductus venosus (dv) and valve of ductus venosus. Axial HREM sections. Note the slender appearance of the valve (arrowhead) in (i) and the plump appearance (arrow) in (j). (k, l) Liver texture of a stage 21 (k) and stage (22)

| Venous malformations in embryos with gene deletions
We explored the usefulness of our reference data using them for identifying abnormalities of the venous system in embryos, which miss both alleles of Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c.

| DISCUSS ION
We present a first, in-depth, digital volume data-based description of the venous system of mouse embryos as it presents itself in the E14.5 developmental stages S21-S23 (Geyer et al., 2017b).
This is based upon examination of a very large number (206)  Usually, images from mounted histological sections are used as reference for diagnosing venous abnormalities in E14.5 embryos (Kaufman, 1992;Theiler, 1989). Since these are 2D, they do not provide 3D information. This, however, is essential for the correct Except for some notable exceptions, the anatomy of the venous system of the mouse is similar to that of humans. Naturally, there are many differences due to specific organ morphology and topology.
The most important larger structural dissimilarities affecting the venous system of mice are as follows: First, a right and left superior vena cava; second, a left-sided azygos vein; third, a confluence of three lung veins dorsal to the left atrium, from which a single pulmonary vein forms that enters the left atrium; and fourth, a different arrangement of liver veins as a consequence of different liver lobulation (Fiebig et al., 2012). Not surprisingly, our results demonstrate that these differences are already established at E14.5.
But, in addition to these general differences between mice and man, there are also differences between adult and unborn mice.  Remodelling of the sinus system, with the sigmoid sinus forming, the primary head sinus vanishing and the transverse sinus becoming symmetric, provides the clearest example of why an accurate staging is a prerequisite for the correct interpretation of the phenotype of E14.5 mice. However, it is also essential to be aware of the various stage-specific and unspecific venous peculiarities in S21 to S23 embryos. These include the architecture of the liver sinusoids and liver vasculature, the connection of vitelline vein topology and physiological gut rotation (Geyer et al., 2017b), the occasional narrowing of the umbilical vein and even the connection of the coronary sinus to the right atrium.
Only by relying on precise descriptive reference data can pathologies such as abnormal liver tissue composition, abnormal ductus venosus valve morphology, intrahepatic shunts between hepatic portal and hepatic venous system be securely defined as malformations. Our study has, for example, identified dual connection of the coronary sinus, a pathology affecting some embryos which was previously unknown. Establishing that a double connection was not a normal but transitory feature at E14.5 required detailed reference data derived from significant numbers of normal embryos at each of the successive stages of E14.5. It remains to be examined in future studies whether this double connection affects haemodynamics is a source for thromboembolic brain ischemia or has any other pathologic relevance. Finally, it has to be emphasised that precise descriptions of the stage dependency of anatomical features not only add to our knowledge of normal development. It is also the basis for the accurate detection of heterochronic events, since simple comparisons of mutants with single-possibly not stage matching-controls might lead to misdiagnoses (Boughner et al., 2018;Geyer et al., 2017b).
The DMDD programme showed that the penetrance of phenotypes varies substantially between different embryos (Wilson et al., 2016a (Reissig et al., 2021).
Whether and which venous variations, features and abnormalities might have a potential to serve as indicators for severe malformations of the cardiovascular or other organ systems remains to be explored.
Our data are based on embryos of the C57BL/6 strain, which is one of the most popular mouse strains of modern biomedicine and had been selected by the IMPC and DMDD as its standard genetic background strain. Thus, the reference data we have presented here may be useful for the broader scientific community working with this mouse strain. To what extent the current data can be transferred to other popular mouse strains remains to be assessed. While there is only scarce information on head veins, some strain peculiarities have been documented that affect the azygos vein and liver anatomy (Biddle et al., 1991;Fiebig et al., 2012).
The results from the DMDD phenotype screen were made publicly available via the DMDD website (www.dmdd.org.uk) as well as the mouse genome informatics site of the Jackson Laboratory (http://www.infor matics.jax.org/). This also applies for the knockout embryos we included in this study. While we merely concentrate on selected venous abnormalities, the full phenotype and an estimation of the penetrance of the single features can be deduced from visiting these pages and some of the lines have already started to be subject of more systematic study (e.g. De Franco et al., 2019;Perez-Garcia et al., 2018;Reissig et al., 2019).

CO N FLI C T O F I NTE R E S T
No conflict of interest declared.

AUTH O R S' CO NTR I B UTI O N S
SHG and WJW designed the study, drafted and revised the manuscript. FP, RW, AG, CT, JKW and TJM created mouse lines and HREM data. LFR and WJW did phenotyping. SHG, BMG and JR created 3D models and did statistics.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.